Viability of Human Arterial Grafts Monitored by Comet Assay.

An analytical method for studying DNA degradation by electrophoresis after cell lysis and visualization of DNA fragments with fluorescent dye, comet assay, was used to evaluate the viability of the endothelial layer of human arterial grafts with the aim of identifying the procedure that will least damage the tissue before cryopreservation. Four groups of samples were studied: cryopreserved arterial grafts that were thawed in two different ways, slowly lasting 2 hours or rapidly for approx. 7 minutes. Arterial grafts that were collected as part of multiorgan procurement with minimal warm ischemia time. Cadaveric grafts were taken as part of the autopsy, so they have a more extended period of warm ischemia. The HeadDNA (%) parameter and others commonly used parameters like TailDNA (%). TailMoment, TailLength, OliveMoment, TailMoment to characterize the comet were used to assess viability in this study. The ratio of non-decayed to decayed nuclei was determined from the values found. This ratio for cadaveric grafts was 0.63, for slowly thawed cryopreserved grafts 2.9, for rapidly thawed cryopreserved grafts 1.9, and for multi-organ procurement grafts 0.68. The results of the study confirmed the assumption that the allografts obtained from cadaveric donors are the least suitable. On the other hand, grafts obtained from multiorgan donors are better in terms of viability monitored by comet assay. Keywords: Arterial grafts, Cryopreservation, Cadaveric, Multiorgan procurement, Viability, Comet assay.

Cognitive sequelae of methanol poisoning involve executive dysfunction and memory impairment in cross-sectional and long-term perspective.

Methanol poisoning leads to lesions in the basal ganglia and subcortical white matter, as well as to demyelination and atrophy of the optic nerve. However, information regarding cognitive deficits in a large methanol sample is lacking. The principal aim of the present study was to identify the cognitive sequelae of methanol poisoning and their morphological correlates. A sample of 50 patients (METH; age 48 ± 13 years), 3-8 months after methanol poisoning, and 57 control subjects (CS; age 49 ± 13 years) were administered a neuropsychological battery. Forty-six patients were followed in 2 years' perspective. Patients additionally underwent 1.5T magnetic resonance imaging (MRI). Three biochemical and toxicological metabolic markers and a questionnaire regarding alcohol abuse facilitated the classification of 24 patients with methanol poisoning without alcohol abuse (METHna) and 22 patients with methanol poisoning and alcohol abuse (METHa). All groups were compared to a control group of similar size, and matched for age, education, premorbid intelligence level, global cognitive performance, and level of depressive symptoms. Using hierarchical multiple regression we found significant differences between METH and CS, especially in executive and memory domains. METHa showed a similar pattern of cognitive impairment with generally more severe executive dysfunction. Moreover, all METH patients with extensive involvement on brain MRI (lesions in ≥2 anatomical regions) had a more severe cognitive impairment. From a longitudinal perspective, we did not find any changes in their cognitive functioning after 2 years' follow-up. Our findings suggest that methanol poisoning is associated with executive dysfunction and explicit memory impairment, supposedly due to basal ganglia dysfunction and disruption of frontostriatal circuitry proportional to the number of brain lesions, and that these changes are persistent after 2 years' follow-up.

[Microglial activation and GFAP astroglial immunopositivity in drug abusers]. / Aktivace mikroglie a GFAP imunopozitivita astroglie u toxikomanu

INTRODUCTION: The neurotoxicity brought about by application of toxic and psychotropic substances is accompanied by an activation of astroglial and microglial cells in the brain. MATERIALS AND METHOD: We investigated clinically 42 patients addicted to psychotropic substances (hospitalised in the Motol Teaching Hospital). The NSE, S1OOB, and manganese concentrations in the blood were measured in the patients. In 14 deceased patients with drug evidence in the hair indicating a chronic abuse of addictive substances, the brain tissue glial cells were immunohistochemically labelled with antibody against CD68 and GFAP. RESULTS: In 8 hospitalised patients, there were increased NSE, S100B values in the blood (p < 0.05). Manganese in the blood was increased (3,03±1,9 µg/l, p < 0,05) in all patients. In deceased persons, the CD68 positivity of microglial cells and neurophagy have been proved. GFAP positive astroglial cells have been evidenced in the gray and white matter. CONCLUSION: The CD68 and GFAP positive glial cells in brain tissue can be a pathomorphological correlate of neurotoxicity in chronic abusers. The neurotoxicity can be monitored with NSE and S100B markers of damaged neuronal cells.

[The origin, distribution and relocability of supravital hemorrhages]. / Vznik, distribuce a mobilita supravitálních hematomu.

The hemorrhage as a result of intravital bleeding is considered, from forensic point of view, as important sign of vital reaction of injury. However, in special cases it must be accepted that hemorrhage occurred after the death. The formation of supravital changes is evident, e.g., in organ donors whose blood circulation and pulmonary ventilation is kept after the brain death. The post-mortem origin of hemorrhages can also be seen in donors of eyeballs after enucleation made before the autopsy at Institute of Forensic Medicine and Toxicology. These hemorrhages are manifested after several hours when eyeballs were removed. Moreover, we observed the origin of hemorrhage in orbit which was caused nor by intravital bleeding neither by direct force. Its origin could not be explained nothing but postmortem propagation of the hemorrhage from the fracture of anterior fossa of the scull base. We did not find information about postmortem origin or relocation of hemorrhage of such extent in the literature. In the frame of knowledge about supravital reaction, this finding is of general importance with forensic impact. The documentation from the scene of death allowed correcting the appreciation of the mechanism of injury and traumatic process from the point of view of foreign culpability which should be considered in such case (e.g., a blow to the orbit with following fall under passing subway train).

[Mass disasters with higher quantity of dead bodies and DVIT concept in the Czech Republic]. / Mimorádné události s vetsím poctem zemrelých a koncepce DVI v CR.

Authors deal with problems related to identification of higher number of dead bodies in cases of mass disaster, organization and DVI (disaster victim identification) teams assignment in the Czech Republic, interoperation with so-called Integrated Rescue System and cooperation between experts by mass disaster solution. The paper comes out from investigation of the research project and the main principles for organization of operations in victims identification of mass disaster are mentioned.

[The correlation of results of breath analysers and laboratory blood examinations of blood-ethanol concentration (Czech and Slovak study)]. / Mezirepubliková Cesko-Slovenská studie korelace výsledku dechových analyzátoru a výsledku laboratorní analýzy pri zjistování hladiny alkoholu v krvi.

The target of this study was to compare the results of breath analysers and "lege artis" laboratory blood examinations when determining alcohol levels. This was then used to determine whether any differences exist between the two methods, and how large these differences are. 610 cases from 11 workplaces in the Czech Republic and Slovakia were analysed. The type of breath analyser was not taken into consideration. All cases had to be in the elimination phase. Difference of time between breath test and blood test were rectified through the use of reverse recomputation. It was detected that only 20.8% of the results of respiratory analyser tests correspond to the detected real alcohol level in blood. The maximum difference when a respiratory analyser measured more than a blood test was 1.34 g x kg(-1). and the maximum difference when the analyse measured less was 1.86 g x kg(-1).

Forensic dentistry--identification from the dentist's point of view.

The paper deals with an identification of missing persons based on a dental documentation. The procedure of the identification is presented in two case reports with a new possibility of electronic imaging called Dental Cross in comparison with classical dental documentation, which is officially used for identification of the missing persons by Interpol.

The image analysis of colour changes of different human tissues in the relation to the age. Part 2: practical applicability.

The method of image analysis of intervertebral disc, Achilles tendon and rib cartilage was applied for assessment of colour changes of these tissues in the relation to the human age. It was proved that colour of tested tissues changes with age which is most obvious on rib cartilage and intervertebral disc, while Achilles tendon does not display important changes. The parameters MeanBlue, MeanSaturation and MeanBrightness are the best for age estimation based on colour analysis.

The image analysis of colour changes of different human tissues in the relation to the age. Part 1. Methodological approach.

The human age for medico-legal purposes is usually estimated from hard tissues like bones and teeth. Only little attention was paid to soft tissues most probably due to the lack of detectable age changes. This study deals with colour changes of human tissue from intervertebral discs, Achilles tendon and rib cartilage in the relation to the age. The image analysis of colour of investigated tissue samples was performed. The values of intensities of channels RGB (MeanRed, MeanGreen, and MeanBlue) and parameters from the IHS system (MeanSaturation, HueTypical, HueVariation, BrightVariation and MeanBrightness) were evaluated. The results confirm that colour changes of some tissues can be used for age estimation.

Oral health status of drug addicts in the Czech Republic.

Over the last decade, illicit drug use has become a very pronounced problem in the Czech Republic. Some branches of medicine such as psychiatry, internal medicine and neurology have tried to confront the situation by joint treatment of addicted patients. Dentistry has so far remained outside this multi-disciplinary care despite the fact that the status of dental and other tissues in the oral cavity influences general health and vice versa. The aim of the survey on the oral health of drug addicts that was conducted in the Czech Republic from 2000 to 2002 was to collect data to help dental professionals contribute to the complex care of drug addicts. The number of addicted persons examined was 400 and the subgroup of drug addicts consisted of 217 subjects (mean age 23.87 yrs, SD 6.70), all being treated in long term hospitals. Among other oral health features observed, the DMFT and CPITN indices are presented in this paper. The results show severe dental and periodontal tissue destruction in young addicts and two case reports are included. The above survey indicates that dental treatment should become a part of standard care for addicted patients in long-term hospitals. Furthermore, if severe oral tissue impairment in young persons whether in dental practice or during autopsy is encountered a drug addiction should be suspected. If so, the treatment of the person or examination of the dead body should accordingly be adjusted.

[Age-related post-translational modification of proteins in human tissue and its use determination of age in forensic medicine. Review]. / Závislost nekterých posttranslacních modifikací proteinu lidských tkání na veku a jejich pouzití pro urcování veku v soudním lékarství. Prehled poznatku.

This article deals with some of posttranslational modification of proteins which are, or could be useful to work out objective and standard methods for age estimation in forensic medicine. From many posttranslational modifications other than racemization, the evaluation of pentosidine, one of the products of nonenzymatic glycosylation, seems to be promising. The enlargement of menu of methods for age estimation is important mainly for forensic sciences when determination of the age of an unknown dead body is necessary. Morphological methods are quite often subjective and charged with errors.

The use of the D-, L- aspartic ratio in decalcified collagen from human dentin as an estimator of human age.

Among the methods dealing with the age estimation, the evaluation of the ratio of the D-, L- form of the aspartic acid in tissues with a low metabolic turnover is considered to be the most precise. We introduced demineralization of the dentin with 0.5 M EDTA adjusted to pH = 7.4. The advantage of such a procedure is that after demineralization we obtained pure insoluble protein (collagen) and soluble noncollagenous proteins in one step. In this study we analyzed insoluble collagen. The amino acids obtained after the hydrolysis were derivatized into TFA isopropyl esters and analyzed by gas chromatography on Chirasil L-Val capillary column. We analyzed human dentin from the lower canines. The correlation coefficient was 0.93 for our set of 71 persons. The result concurred with those of other scientists.

[Determination of the D-and L-forms of aspartic acid in intervertebral disk tissue as an indicator of age]. / Stanovení pomeru D- a L-formy kyseliny asparagové ve tkáni intervertebrálního disku jako ukazatel veku.

Assessment of age according to racemization coefficient of aspartic acid in dentin is considered a reliable method. Proteins which are isolated from dentin are non-collagenous proteins or collagen. As collagen is one of the most frequent proteins in the human body, the question arises whether the D-form of aspartic acid can be assessed also in other tissues and the racemization coefficient used for assessment of age. In the submitted work the author assessed to use the racemization coefficient in the tissue of the intervertebral disk. A lyophilized portion of the annulus fibrosus was hydrolyzed and the amino acids of the hydrolysate were estimated after derivation by gas chromatography on a chiral capillary column. The values of the racemization coefficient indicate limited possibilities to assess age from thus prepared tissue.

[Determination of D- and L- forms of aspartic acid in Achilles tendon tissue as an indicator of age]. / Stanovení pomeru D- a L-formy kyseliny asparagové ve tkáni Achillovy slachy jako ukazatel veku.

The presence of the D-form of aspartic acid was detected in collagen of dentin and the intervertebral disk before and assessment of its racemization coefficient was used for assessment of the age of subjects. Another tissue formed almost solely by collagen type I are tendons. Tendon tissue was not investigated so far with regard to the racemization coefficient and therefore the authors assessed the D-form of aspartic acid in the Achilles tendon. They used lyophilized tendon excisions which were hydrolyzed. Amino acids in the hydrolysate were derived to N-TFA isopropyl esters which were assessed by gas chromatography on a chiral capillary column. In all samples the D-form of aspartic acid was detected. The values of the racemization coefficient suggest the possible use of thus processed tissue with certain limitations.

[Age determination using peptide mapping of non-collagenous proteins in human dentin]. / Moznosti stanovení veku peptidovým mapováním nekolagenních proteinu lidského dentinu.

The submitted paper deals with possibilities of assessment of human age based on findings of stereospecific breakdown of proteins containing D-forms of aspartic acid. The stereospecificity of enzymatic breakdown assumes that after enzymatic hydrolysis peptide breakdown products with different molecular weights--the so-called peptide map--will be formed, depending how many D-aspartyl residues the protein contains. The authors proved in the submitted preliminary study in subjects of different age the formation of breakdown products of different size in non-collagenous proteins of human dentin which was hydrolyzed by protease V8 from Staphylococcus aureus.

A rare case of evisceration.

A case of homicide is described where the body of a young woman was found in the countryside in a state of advanced decomposition. The autopsy revealed that almost all the internal organs were missing, but not due to putrefaction. The thoracic cavity was opened in the professional manner normally used during an autopsy. The remains of the small intestine were tied with strands of hair braided into a thin cord.

Determination of the size of a foreign body in the eye using image analysis of its roentgenogram.

The case of a computer-enhanced image analysis of X-ray pictures of a pellet in the orbit of a man who sustained a shotgun injury to his right eyeball is presented. The man was shot accidentally during a pheasant hunt. Two hunters were shooting simultaneously but they were using three different sizes of pellet--3.0, 3.5 and 4.0 mm. One of the pellets hit a third man in the eye. He sustained serious injury with a resultant loss of sight in this eye despite immediate medical treatment. Unfortunately the pellet could not be removed from the orbit, but it was necessary to establish who was responsible for the injury. The problem lay in determining the specification and size of the pellet at that moment. The problem was solved using computer-enhanced video image analysis of the X-ray pictures of the pellets. The image processor LUCIA G (http(/)/www.lim.cz) was used.

[Age determination by detection of ratios of the D and L forms of aspartic acid]. / Urcení veku stanovením pomeru D,L formy kyseliny asparagové.

Paper concerns evaluation of results of age estimation based on the increase of D-aspartic acid during aging. Samples of dentin from lower canine teeth were used as experimental material. In addition, analytical pitfalls and new possibilities of d-amino acids detection were discussed.

[Age determination by measurement of the ratio of D- and L- forms of aspartic acid. II. Chiral separation of amino acids from human dentin]. / Moznosti urcení veku stanovením pomeru D, L-formy kyseliny asparagové II. Chirální separace aminokyselin z lidského dentinu.

Estimation of relation of D,L-forms aspartic acid has been recently used for an evaluation of age. It represents a relatively new method based on analysis of hydrolyzed dentine. Its amino acids are derived and analyzed by gas chromatography or liquid chromatography on a chiral column.

[Present possibilities of age determination in forensic medicine with emphasis on the importance of measurement of D- and L- forms of aspartic acid. I. An overview]. / Soucasné moznosti urcení veku v soudne-lékarské praxi se zamerením na význam stanovení D, L-forem kyseliny asparagové I. Prehled poznatku.

Evaluation of age of unknown deceased persons belongs to the most important ways to identification. For the time being, morphological methods are used, namely evaluation of age according to Gustafson's method from tooth grindings or by macroscopical estimation of abrasion, transparency of root dentine, alveolar atrophy and number of missing teeth. Evaluation of the data can be influenced by an individual failure and experience showed an age related decrease of precision. Recently, some papers occurred estimating a relation of D, L-forms of aspartic acid which depends on the age with a significant precision.